PROJECT SUMMARY Despite treatment improvements, leukemia-associated mortality is still unacceptably high, underscoring the need for better therapies. Metabolic reprogramming is a hallmark of cancer that enables tumor cells to cope with the metabolic stress imposed by rapid proliferation, and represents an exciting new area of targeted therapy. Therefore, the identification and characterization of the altered metabolic pathways in leukemic cells could lead to novel therapeutic approaches. We recently identified dihydrolipoamide S-succinyltransferase (DLST) as a metabolic ?oncorequisite? enzyme critical for MYC-driven leukemogenesis. We have reported that MYC-dependent T-acute lymphoblastic leukemia (T-ALL) cells reprogram metabolism by stabilizing DLST protein, and rely heavily on its elevated levels for proliferation and survival. Heterozygous loss of dlst in zebrafish significantly delays the onset of Myc-induced T-ALL that resembles a major subtype of human disease with poor prognosis. DLST is a transferase in the tricarboxylic acid (TCA) cycle and mediates oxidative decarboxylation of ?-ketoglutarate (?-KG), which not only serves as the cycle intermediate for glutamine and glucose catabolism, but also an obligatory cofactor for ?-KG-dependent dioxygenases (?-KGDO) to promote their activities. Hence we hypothesize that: DLST protein stabilization in response to metabolic stress enhances the catabolism of glucose and glutamine, and suppresses ?-KGDO activities, thus promoting leukemic cell proliferation and survival. In Aim 1 of this application we will apply genetic, pharmacological and biochemical approaches to determine the mechanisms by which DLST is stabilized in MYC-overexpressing T-ALL cells and identify novel DLST interactors including its E3 ligase(s). The zebrafish T-ALL model will then be utilized to define the role of novel DLST interactors in T-ALL pathogenesis. In Aim 2, we will combine the analyses of the in vivo zebrafish model and human T-ALL cells to identify the biochemical pathways and molecular changes associated with DLST inactivation, as well as functionally characterize the role of DLST-relevant ?-KGDO in T-ALL pathogenesis. In Aim 3, we will investigate the compensatory pathways and targetability of DLST in relapsed or refractory T-ALL by using our newly identified DLST inhibitor and in vivo animal models including murine patient-derived xenografts. The innovation of this application lies in the study of DLST as a novel metabolic ?oncorequisite? enzyme in a physiologically relevant in vivo zebrafish system. Indeed, this innovative system has enabled us to identify MYC and AMP-activated protein kinase as potential regulators for DLST and isocitrate dehydrogenase 2 as its candidate compensatory gene. The significance of this application is that it will deepen our understanding of the reprogrammed metabolic pathways and oncogenic signaling in MYC-driven leukemogenesis, with the long-term goal of uncovering novel therapeutic strategies directed towards DLST-mediated metabolic dependencies in leukemic cells